Researchers in the field of human physiology and pharmacology found TRPV2 (transient receptor potential vanilloid 2) in human in 1999. TRPV2 was presumed to play an essential role in maintaining survival system in various tissues. In particular, TRPV2 is expressed in peripheral sensory nerve fibers which recognize pain. TRPV2 belongs to thermoTRP family (temperature-sensitive transient receptor potential ion channels) that is the pain receptor family recognizing temperature and painful stimuli. Many researchers expect that human pain mechanism will be disclosed by understanding the functions of TRPV2, the pain receptor, and finally the goal of relieving pain will be achieved by the development of a TRPV2 regulator. To examine TRPV2 functions and develop a TRPV2 regulator, a TRPV2 specific activator that only works for TRPV2 without affecting any other TRP receptors is required.
To understand basic techniques used for the development of a TRPV2 specific activator, it is important to understand the characteristics of TRPV2. TRPV2 is an ion channel and its activation makes cations migrate into sensory neurons, causing changes in membrane currents. The changes of membrane currents generate action potential signal and this potential signal is transmitted to the brain where pain is perceived. One of the techniques to measure TRPV2 activation is patch-clamp electrophysiological technique measuring the changes of membrane currents after amplifying thereof. And another technique to measure TRPV2 activation is to measure intracellular calcium level based on the fact that TRPV2 is involved in the migration of cations such as calcium ions. The first technique is superior in sensitivity to the second one, but the second technique is superior in high speed to the first one, so that they are complementary to each other. Such techniques to measure TRPV2 activation can be executed by the support of animal neuron culture technique, cell line culture technique, TRPV2 DNA control and transfection techniques. Various TRPV2 specific activator candidates are administered to TRPV2 over-expressing cells and then TRPV2 activation therein is measured to select a proper TRPV2 activator and determine its capacity.
A TRPV2 specific activator is an essential element to measure TRPV2 activation for further development of a TRPV2 regulator. However, no reports have been made so far in relation to a TRPV2 specific activator. The known TRPV2 activators are 2-APB (2-aminoethoxydiphenyl borate) and cannabinoid compounds. But, 2-APB is not specific to TRPV2 and in fact it can activate other TRP receptors such as TRPV1 and TRPV3, etc, suggesting that it is not very useful. In the meantime, cannabinoid compounds have their own cannabinoid receptor activation activity but have no specificity to TRPV2, making them not a good candidate for a TRPV2 specific activator.
Probenecid has been clinically used as a uricosuric agent for hyperuricemia which is a cause of gout and used to be co-treated with an antibiotic so as to inhibit the discharge of the antibiotic to increase the blood level of the antibiotic. The mechanism of probenecid based on the above two characteristics is to interrupt organic anion transporters carrying uric acid or antibiotics in the kidney. It has also been reported that probenecid interrupts such ion channel as CFTR (cystic fibrosis transmembrane conductance regulators). Such interrupting activities have nothing to do with the TRPV2 activation in sensory nerve.
Therefore, the present inventors constructed transformants expressing TRP and treated them with probenecid and other chemicals known as TRP activators, followed by comparison of the results. As a result, the inventors completed this invention by confirming that probenecid activated TRPV2 specifically and thus it can be effectively used for the screening of a TRPV2 activation blocker.